Cytochrome P450 3A4-Mediated Oxidative Conversion of a Cyano to an Amide Group in the Metabolism of Pinacidil
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- 作者：Zhoupeng Zhang ; Ying Li ; Ralph A. Stearns ; Paul R. Ortiz de Montellano ; Thomas A. Baillie ; and Wei Tang
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：February 26, 2002
- 年：2002
- 卷：41
- 期：8
- 页码：2712 - 2718
- 全文大小：89K
- 年卷期：v.41,no.8(February 26, 2002)
- ISSN：1520-4995
文摘
The conversion of nitriles to amides is generally considered to be a hydrolytic process thatdoes not involve redox chemistry. We demonstrate here that cytochrome P450 (CYP) is responsible forthe conversion of the cyano group of pinacidil to the corresponding amide. The reaction in human livermicrosomes was NADPH-dependent and was nearly completely inhibited by an anti-CYP3A4 antibody.Incubations of pinacidil with recombinant CYP enzymes confirm that CYP3A4 is the principal catalystof this reaction. The kinetics of pinacidil amide formation by CYP3A4 yielded an apparent Km of 452 ±33 
M and kcat of 0.108 min-1 (kcat/Km = 0.238 mM-1·min-1). Incubation of pinacidil with CYP3A4 inthe presence of 18O2 or H218O showed that the amide carbonyl oxygen derived exclusively from molecularoxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubationswere carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explainedby a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe3+-O-O-) on the carbonatom of the -C
N triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed bycleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe3+-O-O-intermediate to a -C=N 
-bond in a P450 system resembles the analogous reaction catalyzed by thenitric oxide synthases.
M and kcat of 0.108 min-1 (kcat/Km = 0.238 mM-1·min-1). Incubation of pinacidil with CYP3A4 inthe presence of 18O2 or H218O showed that the amide carbonyl oxygen derived exclusively from molecularoxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubationswere carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explainedby a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe3+-O-O-) on the carbonatom of the -CN triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed bycleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe3+-O-O-intermediate to a -C=N -bond in a P450 system resembles the analogous reaction catalyzed by thenitric oxide synthases.